Converting biopolymers to extracellular matrix (ECM)-mimetic hydrogel-based scaffolds has provided invaluable opportunities to design in vitro models of tissues/diseases and develop regenerative therapies for damaged tissues. Among biopolymers, gelatin and its crosslinkable derivatives, such as gelatin methacryloyl (GelMA), have gained significant importance for biomedical applications due to their ECM-mimetic properties. Recently, we have developed the first class of in situ forming GelMA microporous hydrogels based on the chemical annealing of physically crosslinked GelMA microscale beads (microgels), which addressed several key shortcomings of bulk (nanoporous) GelMA scaffolds, including lack of interconnected micron-sized pores to support on-demand three-dimensional-cell seeding and cell-cell interactions. Here, we address one of the limitations of in situ forming microporous GelMA hydrogels, that is, the thermal instability (melting) of their physically crosslinked building blocks at physiological temperature, resulting in compromised microporosity. To overcome this challenge, we developed a two-step fabrication strategy in which thermostable GelMA microbeads were produced via semi-photocrosslinking, followed by photo-annealing to form stable microporous scaffolds. We show that the semi
Objective: In this work we propose a method for producing engineered human derived three-dimensional neuronal assemblies coupled to Micro-Electrode Array (MEA) substrates for studying the electrophysiological activity of such networks. Approach: We used biocompatible chitosan microbeads as scaffold to build 3D networks and to ensure nutrients-medium exchange from the core of the structure to the external environment. We used excitatory neurons derived from human-induced Pluripotent Stem Cells (hiPSCs) co-cultured with astrocytes. By adapting the well-established NgN2 differentiation protocol, we obtained 3D engineered networks with good control over cell density, volume and cell composition. We coupled the 3D neuronal networks to 60-channel Micro Electrode Arrays (MEAs) to evaluate and monitor the functional activity of the neuronal population. In parallel, we generated two-dimensional neuronal networks to compare the results of the two models. Main results: 3D cultures were healthy and functional up to 42 Days In Vitro (DIVs). From the structural point of view, the hiPSC derived neurons were able to adhere to chitosan microbeads and to form a stable 3D assembly thanks to the connections among cells. From a functional point of view, neuronal networks showed spontaneous activity after a couple of weeks. We monitored the functional electrophysiological behavior up to 6 weeks and we compared the network dynamic with 2D models. Significance: We presented for the first time a method to generate 3D engineered cultures with human-derived neurons coupled to MEAs, overcoming some of the limitations related to 2D and 3D neuronal networks and thus increasing the therapeutic target potential of these models for biomedical applications.
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